FIG. 4.

p73 is sumoylated only at lysine 627. (A) H1299 cells were transiently transfected with either HA-TAp73α or its mutant HA-TAp73α(K627R) in combination with Flag-SUMO-1 and Flag-PIAS-1 expression vectors. Cell lysates were subjected to Western blot analysis performed with anti-HA antibody (upper panel). The same blot was reprobed with anti-PIAS-1 antibody (middle panel) and with an antiactin antibody (lower panel). The p73 mutant HA-TAp73α(K627R) cannot be sumoylated regardless of the coexpression of SUMO-1 and/or PIAS-1 (lanes 6 to 8). This mutant p73 is still capable of binding PIAS-1. Asterisks indicate sumoylated p73. IB, immunoblot. (B) HA-TAp73α and HA-TAp73α(K627R) expression vectors were transfected in H1299 cells together with the Flag-PIAS-1 expression vector. Cell extracts were immunoprecipitated (IP) with anti-Flag antibody and subjected to Western blot analysis performed with anti-HA antibody (upper panel). The same blot was reprobed with anti-HA antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gels (lanes 1 to 6). (C) The PIAS-1 mutant C350S is no longer capable of sumoylating p73. H1299 cells were transfected with HA-p73α in combination with Flag-SUMO-1 and wild-type Flag-PIAS-1 or mutant Flag-PIAS-1(C350S). Cells were subjected to Western-blot analysis performed with anti-HA antibody (upper panel). The same blot was reprobed with anti-PIAS-1 antibody (middle panel) and with an antiactin antibody (lower panel). (D) The PIAS-1 mutant (C350S) is still capable of binding p73. HA-p73α expression vectors were transiently transfected in H1299 cells, together with mutant Flag-PIAS-1(C350S). Cell extracts were immunoprecipitated with anti-Flag antibody and were subjected to immunoblot analysis with anti-HA antibody (upper panel). The same blot was reprobed with anti-Flag antibody (lower panel). Aliquots of total cell extract from unprocessed cells were also loaded on the gel (lanes 1 to 4).